Understanding the Immune Response to Glycans of Schistosoma mansoni Pubblico

Abstract

Glycans are ubiquitous at the interface between pathogens and host immunity, both as stimulators of innate immunity and targets of adaptive immunity. Schistosomiasis, caused by infection with parasitic helminthes of the Schistosoma spp., is a Neglected Tropical Disease that poses an enormous public health burden to the global community, and which features a prominent anti-glycan antibody response. The glycans of this parasite could provide a wealth of possible diagnostic and vaccine targets; however, whether anti-glycan antibodies play a protective role in this infection is not known, and there are no precedents for design of a vaccine targeting eukaryotic glycans. We sought to better understand how glycan antigens similar to those of S. mansoni elicit immunity by characterizing the immune response to differentially-linked neo-glycoconjugates (LNnT-BSA) and recombinantly-engineered, poly-LDNF producing cells. We then explored the role of anti-glycan antibodies in schistosomiasis by comparing the anti-glycan reactivity of protected with non-protected animal hosts, children from endemic areas, and resistant and susceptible adults on glycan microarrays. These studies demonstrated the importance of mode of presentation for glycan antigens in immunization, the success of recombinantly-engineered cells as a novel platform for presenting glycan antigens, the exquisite specificity of anti-glycan antibodies, and the extremely complex and still enigmatic relationship of anti-glycan antibodies with schistosomiasis resistance in animals and humans. This work should encourage and inform future studies on methods of incorporating glycans into vaccines and on the role of anti-glycan antibodies in schistosomiasis and other helminth infections.

Table of Contents

Introduction xvi

1.1 The burden of schistosomiasis and areas of need 1

1.1.1 Global burden of helminthiases and schistosomiasis 1

1.1.2 Need for innovation to reduce the burden of schistosomiasis 2

1.1.3 Why study anti-glycan immune responses to schistosomes? 3

1.2 The interface of schistosomes and their hosts 4

1.2.1 S. mansoni life cycle and biology 4

1.2.2 S. mansoni immunology 6

1.3 Schistosome glycoconjugates in host-helminth interactions 10

1.3.1 Structural features of schistosome glycoconjugates 15

1.3.2 The role of glycans in schistosome biology 20

1.3.3 The role of glycans in innate immunity to schistosomiasis 22

1.3.4 The role of glycans and anti-glycan antibodies in adaptive immunity to schistosomiasis 26

1.3.5 Evidence that αGAbs can mediate destruction of helminthes and protection from infection 28

1.4 The current state of schistosomiasis diagnostics and vaccine development, and opportunities for improvement through glyco-technology 31

1.4.1 Recent progress in diagnostics 31

1.4.2 Recent progress in immune correlates and vaccine development 33

1.5 Recent developments in glyco-technology 37

1.5.1 Glycan synthesis and glycoconjugate production 37

1.5.2 Glycan microarray screening 37

1.5.3 Genomics and glycosyltransferase expression, characterization, regulation 41

1.6 Major questions facing the schistosomiasis field 41

1.7 Our project aims 43

Chapter 2. An intact reducing-end sugar improves the specificity of the immune response to lacto-N-neotetraose-BSA glycoconjugates 45

2.1 Introduction 45

2.2 Materials and Methods 49

2.2.1 Preparation of conjugate vaccines 49

2.2.2 Immunizations 51

2.2.3 Preparation and analysis of glycan microarrays 51

2.2.4 ELISA 52

2.3 Results 53

2.3.1 Synthesis and characterization of LNnT-BSA glycoconjugate vaccines 53

2.3.2 Closed-ring linkage induces greater glycan specificity than open-ring linkage in rabbits 55

2.3.3 Mice immunized with closed-ring LNnT-BSA generate a muted glycan-specific response 66

2.4 Discussion and Conclusions 67

2.5 Acknowledgements 71

Chapter 3. Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite 72

3.1 Introduction 72

3.2 Materials and Methods 76

3.2.1 Materials. 76

3.2.1.1 Cell culture reagents 76

3.2.2 Methods 78

3.3 Results 89

3.3.1 Recombinantly engineered Lec8 cells express surface-bound poly-LDN and poly-LDNF 89

3.3.2 Recombinantly expressed poly-LDN and -LDNF are cross-reactive with schistosome antigens. 93

3.3.3 Lec8-GT and -GTFT cells induce IgM and IgG antibodies specific for LDN and LDNF glycans. 99

3.3.4 Antibodies to parasite glycans discriminate among very similar presentations of the epitopes. 102

3.3.5 Lec8-GTFT cell antiserum binds to schistosome glycans. 106

3.4 Discussion 108

3.5 Acknowledgements 113

Chapter 4. Schistosoma mansoni-infected Brown Rats Target Core Xylose/Core α3 Fucose Epitopes during a Protective Response 115

4.1 Introduction 115

4.2 Materials and Methods 117

4.2.1 Antibodies 117

4.2.2 Animal infections 117

4.2.3 Array binding assays 118

4.2.4 Isolation of Schistosoma mansoni life stages and preparation of parasite lysates 119

4.2.5 SDS-PAGE and Western blots 121

4.2.6 ELISA 122

4.3 Results 123

4.3.1 Secondary-infected rat sera has broad anti-glycan targeting of Lewis X and poly-N-acetyllactosamines (poly-LN) on the Consortium for Functional Glycomics Array 123

4.3.2 Rats, but not mice, target core xylose/core α3 fucose on the Defined Schistosome-Type Array 131

4.3.3 Infected rat sera binds CX/CF on HRP 134

4.3.4 Rat antisera, mouse antisera and anti-HRP target several overlapping molecular species on Western blot 137

4.3.5 HRP blocks reactivity of rat antisera with select parasite moieties 139

4.3.6 Rat and mouse IgG against larval and adult lysates is predominantly to periodate-sensitive epitopes 140

4.4 Discussion 141

Chapter 5. Anti-glycan antibodies in a population of occupationally S. mansoni-exposed resistant and susceptible humans 147

5.1 Introduction 147

5.2 Materials and Methods 150

5.2.1 Study population and plasma samples. 150

5.2.2 Consortium for Functional Glycomics Array (CFG). 150

5.2.3 Defined Schistosomal-type Array (DSA) and Natural Egg Array. 151

5.2.4 SEA ELISA with/without periodate treatment. 151

5.3 Results 152

5.3.1 Reactivity of resistant and susceptible plasmas to egg glycans 152

5.3.2 Pooled resistant and susceptible plasma on the Consortium for Functional Glycomics (CFG) array. 153

5.3.3 Pooled resistant and susceptible plasma on schistosome glycan arrays. 154

5.3.4 Isotype composition of individual resistant and susceptible plasmas on the egg array. 157

5.3.5 Patterns of susceptible, resistant and changing individuals on the DSA over time. 160

5.4 Discussion 167

5.5 Acknowledgements 169

Chapter 6. Patterns of reactivity to Schistosoma mansoni egg glycan antigens in a population of treatment-naïve Kenyan school children 170

6.1 Introduction 170

6.2 Materials and Methods 174

6.2.1 Study Population. 174

6.2.2 Glycan microarrays 174

6.2.3 Preparation of ELISA antigens 176

6.3 ELISA antigen coating and in-plate periodate treatment 176

6.4 ELISA 177

6.4.1 Data analysis 178

6.5 Results 180

6.5.1 Children in S. mansoni-endemic areas respond with highly variable patterns of IgG and IgM to parasite-associated glycan antigens 180

6.5.2 The response to periodate-resistant soluble egg antigens becomes more dominant as children age 184

6.5.3 Antibodies to select glycan antigens decrease as children age 187

6.5.4 Anti-glycan antibodies in infected children belong to a variety of subclasses 191

6.5.5 Rates of positive reaction to glycoprotein antigens 194

6.5.6 Relationship with infection intensity 194

6.6 Discussion 194

6.7 Acknowledgements 199

Chapter 7. Discussion 200

7.1 Part I: Eliciting immunity to parasite glycans: From synthetic glycoconjugates to novel recombinant technologies 200

7.2 Part II: Investigating the role of anti-glycan antibodies in schistosome infection 205

7.3 Conclusions and future directions 212

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Parasitology Health Sciences, Immunology
Parola chiave	Glycan Anti-glycan antibody Schistosomiasis Immunization
Committee Chair / Thesis Advisor	Cummings, Richard, Emory University
Committee Members	Rengarajan, Jyothi, Emory University Moreno, Alberto, Emory University Jacob, Joshy, Emory University Steinhauer, David, Emory University

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